Activation of T lymphocytes and the subsequent signal transduction events mediated through the TCR/CD3 molecule complex are important events in the immune response against tumors and in autoimmune diseases. Protein tyrosine kinases are intimately involved in T cell signaling through TCR/CD3. However, the role of heterotrimeric G proteins in this process is not clearly established. Therefore, in the present application we are proposing to study the involvement of G proteins in T cell signal transduction. We have observed that TCR/CD3 ligation induces the interaction between G proteins and the CD3 chain. We propose to study this interaction by the use of chimeras and mutants of the intracytoplasmic tail of the CD3 epsilon chain in an attempt to define the critical sites involved in this physical association. We will also use synthetic peptides representing specific sites on G alpha subunits, as well as point mutants of G alpha proteins to determine whether they can interfere with the G alpha-CD3 epsilon association. Co-immunoprecipitation and western blotting analyses with specific antibodies, and CD3 epsilon-GST fusion proteins will be additional tools utilized in order to address this issue. We have also observed that upon TCR/CD3 perturbation, G proteins associate with tyrosine kinases and they regulate their function. We proposed to study this process by focusing on the specific tyrosine kinase EMT, a Tec family member. We will examine the TCR/CD3-mediated physical association and the functional consequences of EMT - G beta and EMT-CD3 epsilon interactions. We will utilize truncation mutants of EMT in order to determine sites that are important for these interactions. The association of src kinases fyn and lck with the EMT-G beta-CD3 epsilon complex will be assessed, and the consequences of this association on src kinase activity will be examined. The effects of G protein function-deficient mutants on the above processes will be also determined. The above analyses will be extended to human thymocytes and peripheral T cells in order to better establish the biological relevance of these events.